Project Summary Melanoma is the deadliest skin cancer and is estimated to cause 9,730 deaths this year. Up to 50% of these deaths will be due to brain metastasis, a particularly aggressive form of melanoma that significantly worsens prognosis. Treatment options for melanoma brain metastasis are limited and historical survival times range from 4-9 months. This is mainly because the brain is a notoriously difficult site to treat, both due to the blood-brain- barrier which prevents penetration of drugs into the brain, its inability to accommodate significant swelling, and the drastic complications which would result from damaging surrounding brain tissue. Recently some patients with melanoma brain metastases have shown clinical responses to immune checkpoint inhibitors, suggesting that immunotherapy may be a new, promising avenue of treatment. While these results indicate that the immune system can mount a significant response to melanoma tumor growth in the brain, a more thorough understanding of the nature of these immune interactions is needed. Our laboratory has established a model of immune- mediated tumor regression in the skin using an immunogenic murine melanoma cell line. Preliminary data shows that when this cell line is injected into the arterial circulation to model metastasis, it reliably forms brain tumors that also regress. Using our novel mouse model, this study will investigate the hypothesis that the composition of the immune microenvironment in melanoma brain metastases determines the extent and quality of the local immune response. Aim 1 will characterize the tumor immune microenvironment of melanoma brain metastases in mice using live animal and ex vivo fluorescent imaging, as well as extensive histological analysis. These results will then be compared to the immune microenvironment of anti-PD1 treated and untreated melanoma brain metastases in human tissue samples to establish the clinical relevance of our model. Aim 2 will examine which immune populations and effector mechanisms are required for successful elimination of brain metastases in mice using depletion studies and knock out strains. Completion of this proposal will elucidate how the tumor immune microenvironment affects melanoma growth in the brain, a crucial step in the development of better immune therapies.